1. Field of the Invention
This invention relates generally to bacterial diseases and more specifically to combinations of compounds that enable therapeutic control of bacterial infections at doses of the compounds much lower than either of the compounds administered alone, especially inhibitors of MurB in combination with β-lactam antibiotics.
2. Background Information
The few available distinct classes of antimicrobial compounds limit the scope for single and combination drug treatment of bacterial infections, including infections involving antibiotic-resistant bacteria. Antibacterial chemotherapy research has therefore focused on the discovery of novel targets for new antibacterial development. An alternative approach to the discovery of new antibacterial compounds is the discovery of antibiotic synergists. Synergism in antimicrobial therapy is well known and is used to describe supra-additive activity of antibiotics used in combinations. For example, in the treatment of bacterial infections combinations such as penicillin or ampicillin and streptomycin or gentamycin have been shown to have a supra-additive effect against enterococci infections. Similarly, carbenicillin or ticarcillin combined with an aminoglycoside such as gentamycin or tobramycin exhibit a synergistic effect in the treatment of Pseudomonas aeruginosa infection. Combined therapy using streptomycin together with tetracycline is more effective in the therapy of brucellosis than either agent alone, and a mixture of chloramphenicol plus a sulfonamide is more effective against meningitis due to Haemophilus influenzae. 
One method, which is used to predict the efficacy of antibacterial agents is described by Scribner et. al., (1982, Antimicrobial Agents and Chemotherapy 21(6):939-943) and in Goodman & Gilman (1980, The Pharmacological Basis of Therapeutics, Sixth Edition, pp. 1097-1098) and is referred to as the checkerboard assay. This assay involves serial two-fold dilutions of the antibiotics individually and in combination in broth, which is then inoculated with the microorganism to be tested. After incubation, the minimum inhibitory concentration (MIC) of each drug used individually and in combination is determined (N.B., the MIC is the lowest concentration of the drug that inhibits growth in the medium). Synergism is indicated by a decrease in the MIC of each drug when used in combination. Antagonism is indicated by an increase in the MIC of either or both drugs when used in combination.
In the above examples, synergism is discovered through the empirical testing of pairs of antibiotics. Synergists may also include compounds that inhibit pathogens capacity to inactivate the antibiotic through metabolic detoxification, compounds that inhibit cellular pumps that export the antibiotic, and compounds that otherwise decrease the minimal inhibitory concentration (MIC) of the antibiotic. Development of a combination containing Amoxycillin and Clavulanic acid is the best example to illustrate such efforts. Amoxycillin is an amino-penicillin and is degraded by beta-lactamase producing bacteria. Clavulanic acid was discovered to inhibit the activity of beta-lactamase but is devoid of antibacterial activity of its own (Reading C & Cole M, 1977. A beta lactamase inhibiting beta-lactam from Streptomyces clavuligenus; Antimicrob Agents Chemother, 11, p-852-857; Reading C, Farmer T & Cole, M 1983, The Beta lactamase stability of Amoxycillin with beta lactamase inhibiting Clavulanic acid, J. Antimicrob Chemother., 11, p-27-32; Todd P A & Benfield P 1990. Amoxycillin/Clavulanic acid, an up-date of its antibacterial activity, pharmacokinetic properties & Therapeutic use, Drugs 39, p-264-307).
In the case of clavulanic acid, the importance of beta-lactamases in imparting resistance to beta-lactam antibiotics was well understood. This understanding allowed targeted development of beta-lactamase inhibitors.
There may be other processes important in limiting the effectiveness of any particular antibiotic. An understanding of the different processes can result in the identification of targets for synergist development much like the case of clavulanic acid. Therefore, it would be useful to have a general way of finding processes that limit antibiotic activity so that targeted synergist development could occur.
Bacterial cell wall biosynthesis is an essential process in bacteria in which peptidoglycans are produced inside the bacteria, transported to the outer membrane, and cross-linked together to form the cell wall. Important therapeutic antibiotics, including penicillins and cephalosporins, inhibit the cross-linking process catalyzed by penicillin-binding proteins. FIG. 1 is a diagram of the process. Of particular interest is the step catalyzed by the MurA enzyme (also known as UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7), which is encoded by murA gene and inhibited by fosfomycin. Also of interest is the next step catalyzed by the MurB enzyme and encoded by the murB gene UDP-N-acetylenolpyruvylglucosamine reductase, EC 1.1.1.158).
The process occurring outside the cell membrane, (transglycosylation and transpeptdation) cross-links peptidoglycan monomers. These reactions are catalyzed by penicillin-binding proteins (PBPs), which are the target sites of the penicillin and cephalosporin antibiotics. There are multiple PBPs in bacteria, each species having its own assortment. The PBPs occur in a small number of copies per cell, from a few hundred to one or two thousand and include low-molecular weight (low-Mr) PBPs, high-Mr PBPs, and β-lactamases. All three classes have active-site serine residues important in their catalytic function. Penicillin and cephalosporins act as suicide substrates that inactivate PBPs, thus inhibiting the final steps of cell wall biosynthesis. β-lactamases are structurally related to the PBPs with active-site serines that act on penicillins and cephalosporins. However, in the case of the β-lactamases, the interaction with the penicillin or cephalosporin results in the hydrolysis of the antibiotic, ruining their ability to interact with PBPs.
There are many studies in the literature concerning the combination of fosfomycin with cephalosporins and penicillins. Alvarez et al. (1985) reported synergism was observed in 66% of strains with fosfomycin-cefamandole and in 46% of strains with fosfomycin-methicillin. The synergy reported in this study was defined if the minimum inhibitory concentrations (MICs) of both drugs decreased by one-fourth. Utsui et al (1986) reported on in vitro and in vivo antibacterial activities of cefinetazole and cefotaxime alone and in combination with fosfomycin against methicillin- and cephem-resistant strains of Staphylococcus aureus. This study showed the one-fourth reduction in MIC for both drugs as a measure of synergy. This study also demonstrated accelerated killing of bacteria in the combination treatments, another form of synergy.
The study of Utsui et al. included detection of fosfomycin-induced specific PBP profiles.
The levels of several of the PBPs were reduced significantly upon exposure to fosfomycin. Since cephalosporins act by inactivating PBPs, the researchers concluded a reduction in PBPs would make the cephalosporins more effective by reducing the number target proteins to inhibit. They further speculated that peptidoglycan monomers are inducers of PBP transcription. Inhibition of peptidoglycan biosynthesis by fosfomycin would reduce the levels of peptidoglycan, thus inhibiting transcription of PBPs.
Genetic studies on the interaction of peptidoglycan biosynthesis enzymes and beta-lactamase inhibitors demonstrate that interference in murE or murF in methicillin-resistant Staphylococcus aureus have increased sensitivity to the beta-lactam oxacillin (Sieradzki et al., 1997; Sobral et al., 2003; Gardete et al., 2004). The implications of these studies are that MurE and MurF activity is necessary for the expression of beta-lactam resistance. It is therefore logical that inhibitors of MurE or MurF could also increase the sensitivity of methicillin-resistant S. aureus to oxacillin and other beta-lactam antibiotics.
None of these studies anticipate that a chemical inhibitor of peptidoglycan biosynthesis enzymes would increase the sensitivity of non-resistant bacteria to beta lactam antibiotics.
Antisense has been useful in identifying targets in bacteria for new antibiotics. There are many publications demonstrating the use of antisense to discover such targets and to use the antisense clones as sensitized cells to detect inhibitors of the antisense target.
The concept behind these inventions are (1) antisense expression can differentiate genes that are specifically related to ‘essential’ to bacteria, thus defining a set of targets for use in antibiotic discovery and (2) antisense expression can result in cells sensitized to compounds specifically inhibiting the protein corresponding to the mRNA targeted by the antisense. This latter use of antisense can result in the use of sensitized cells in finding target-specific inhibitors from a chemical library or a natural product collection.
None of these references describe the use of antisense to identify targets for synergist discovery.
Useful synergistic combinations of antibiotics can be discovered through systematic testing of pairs of antibiotics, through targeted research for compounds that decrease antibiotic metabolism by the pathogen, or through random or rational screening of chemical entities or extracts with specific antibiotics.
Antisense has been used to discover essential genes and to develop tools for discovery of novel antibiotic compounds.